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研究生:鄧明偉
研究生(外文):Deng, Ming-Wei
論文名稱:不同惡性度腎臟清細胞癌中促發炎細胞激素與趨化激素之探討
論文名稱(外文):Pro-inflammatory cytokines and chemokines in renal clear cell carcinoma with different malignancies
指導教授:孫光蕙
指導教授(外文):Sun, Kuang-Hui
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:63
中文關鍵詞:腎細胞癌促發炎細胞激素趨化激素腫瘤細胞侵略試驗
外文關鍵詞:Renal cell carcinomaPro-inflammatory cytokinechemokineMatrigel invasion assay
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中文摘要
  促發炎細胞激素 (pro-inflammatory cytokines) 與趨化激素 (chemokines),是參與急性發炎反應的因子,會增加血管通透性、吸引活化淋巴球與巨噬細胞。在腎細胞癌病患,發現血清中促發炎細胞激素 (IL-1b、IL6及TNF-a) 與趨化激素 (CXCL4) 濃度上升與病程惡化有關聯性,為了進一步探討之間的關連性,本篇選用兩株不同惡性度腎清細胞癌細胞株,分別為 A498 (Grade IV) 與 786-O (Grade II),探討這些分泌型因子扮演的角色。
  於惡性度高之 A498 腫瘤細胞中 IL-1b、IL-6、IL-12p35、TNF-a、CCL2、HIF-1a、MMP2 及 LIMK-1 訊息核醣核酸表現量較高;CXCL5、MMP1 及 MMP9 則在 786-O 腫瘤細胞表現量較高;VEGF、TGF-b 及 LIMK-2於兩株細胞表現量相同。腫瘤細胞侵略能力分析中,A498 腫瘤細胞具有較高的爬行能力,且 A498 腫瘤細胞培養液可促進 786-O 腫瘤細胞爬行能力增加 14 倍,推測其細胞培養液所含分泌型因子所造成的影響。利用免疫去除法分析 A498 腫瘤細胞培養液中的 MMP2、IL-1b、TNF-a 之影響,發現 A498 培養液促進 786-O 腫瘤細胞侵略能力分別下降 14%、19%、35%;去除 CCL2 及 IL-6 無統計上的差異;去除IL-1b 和 IL-6,可抑制 786-O 腫瘤細胞爬行 34%;共同去除IL-1b、IL-6 與 TNF-a,則使 786-O 腫瘤細胞侵略能力下降 43%。進一步以 786-O 細胞培養液外加不同濃度 TNF-�� 進行細胞侵略分析,發現隨著 TNF-�� 濃度上升,786-O 腫瘤細胞爬行能力隨之增加,確認TNF-a對 786-O 細胞侵略影響最大。收取不同grade的腎臟清細胞癌細胞培養液以 ELISA 分析 TNF-�� 的產量,在 grade IV的細胞株 (A498、ACHN) 分泌 TNF-a�n量較高 (56.7、41.9 pg/ml),反之在 grade II (786-O) 和 III (Caki-1) 分泌 TNF-a 量較低 (9、6.5 pg/ml),而此四種細胞之腫瘤細胞侵略能力隨 Grade 上升侵略能力也增加,顯示高惡性度腫瘤侵略能力與 TNF-a分泌量呈正相關性。
  上述結果可知,以免疫去除法得知在惡性度高的腎清細胞癌 A498 腫瘤細胞培養液中,IL-1b、IL-6、TNF-a、MMP2促使惡性度低的 786-O 細胞侵略能力增加,表現惡性度高的特徵。其中 TNF-�� 增加腫瘤細胞爬行影響最大,且與濃度呈正相關。此項發現將可應用在臨床診斷上,區分惡性度高低或用於開發新型治療的方向。
Abstract
Pro-inflammatory cytokines and chemokines are considered to involve in tumorigenesis by facilitating tumor proliferation and metastasis. In renal cell carcinoma, levels of IL-6, IL-1�� and TNF-�� in serum were significantly higher in the metastasis group and correlated with tumor size. However, little is known about these cytokines in migration and progression of renal clear cell carcinoma (ccRCC). This study was designed to determine the roles of cytokines and chemokines in human ccRCC cells with different malignancies, A498 cells with higher malignancy and 786-O cells with lower malignancy. Expressions of IL-1��, IL-6, TNF-��, HIF-1��, MMP2, IL-12p�����z LIMK-1, and CCL2 were significantly elevated in A498 cells whereas CXCL5, MMP1 and MMP9 expressions were up-regulated in 786-O cells. In Matrigel invasion assay, the number of A498 invaded cells attracted by culture medium containing 10% FBS was 3.1-fold higher than that of 786-O cells. The invasiveness of 786-O cells attracted by A498 conditioned medium (798 cells/field) was markedly raised compared to 786-O conditioned medium (14 cells/field). However, after the immunodepletion of TNF-�� from A498 conditioned medium, the 786-O cell invasion attracted by A498 conditioned medium was inhibited (35%). This inhibition rate was significantly higher than those at the removal of IL-1�� (19%) or MMP2 (14%). Eliminating both IL-1�� and IL-6 from A498 conditioned medium also led to a 34% reduction of 786-O invaded cells. A significant increase in the reduction rate was observed in 786-O cells attracted by A498 conditioned medium without IL-1��, IL6, and TNF-�� (43%). 786-O cells penetrated to the lower surface were significantly promoted by TNF-�� as a dose-dependent manner. Moreover, to evaluate the correlation between TNF-�� secretion and cell invasion was determined by ELISA and Matrigel invasion assay. A498 and ACHN cells, ccRCC cell lines with grade IV, produced high level of TNF-�� (56.7 and 41.9 pg/ml) correlated with more invading cells (262 and 179 cells/field) whereas Caki-1 cell with grade III and 786-O cell with grade II produced low level of TNF-�� (6.5 and 9 pg/ml) correlated with less invading cells (122 and 76 cells/field). These findings indicate that pro-inflammatory cytokines, especially TNF-��, may enhance cell invasion, and finally promote the disease progression. Furthermore, understanding the roles of pro-inflammatory cytokines in the process of tumor promotion will assist in the development of novel cancer therapeutics.
中文摘要 P.02-P.03
英文摘要 P.04-P.05
第一章、導論 P.06-P.12
第二章、材料與方法 P.13-P.36
第三章、結果 P.37-P.41
第四章、討論 P.42-P.45
第五章、參考文獻 P.46-P.56
第六章、結果圖表 P.57-P.63
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